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Background: Macrophage migration inhibitory factor (MIF) is a cytokine capable of stimulating inflammatory cytokine and matrix metalloproteinase production from macrophages and synovial fibroblasts, which leads to persistent inflammation and bone degradation, two of the major pathological processes in rheumatoid arthritis (RA). The aim of this study was to evaluate the association of MIF promoter polymorphisms (−794CATT5‐8rs5844572 and −173G > C, rs755622), circulating MIF levels, and mRNA expression with RA susceptibility and disease activity. Methods: A case–control study was conducted in 200 RA patients and 200 control subjects (CS) from Southern Mexico. Genotyping was performed by conventional PCR and PCR‐RFLP methods. MIF mRNA expression was quantified by real‐time PCR and MIF serum levels were determined by an ELISA kit. Results: The 7,7 (−794CATT5‐8) and −173CC (−173G > C) genotypes were associated with higher disease activity in RA patients. MIF serum levels were increased, and MIF mRNA expression was reduced in RA patients as compared to CS. In addition, RA patients with moderate disease activity had higher MIF levels than those with low disease activity. The −794CATT5‐8 and −173G > C MIF polymorphisms were not associated with RA susceptibility. Conclusion: These results suggest an important role of MIF polymorphisms and MIF serum levels with disease activity in RA. [ABSTRACT FROM AUTHOR]